Endothelin-1 (ET-1), a peptide comprising 21 amino acids, is the strongest known vasoconstrictor. Since its discovery in 1988 by Yanagisawa et al. [27; numerical data in square brackets relate to the attached list of references], biosynthesis, mode of action and association with diseases have been comprehensively investigated and summarized in topical review articles [1, 7, 12, 17, 24]. There are three isoforms of endothelin which are coded by different genes (endothelin-1, endothelin-2, endothelin-3) of which endothelin-1 is present in the greatest concentrations and is the most effective. Endothelin-1 is synthesized in endothelium cells, in the lung, in the heart, in the kidney and in the brain. The primary translation product of the human endothelin-1 gene is a peptide comprising 212 amino acids, preproendothelin-1 (SEQ ID NO: 1). In the secretion process, a short N-terminal signal sequence (amino acids 1-17) of the preproendothelin is removed by the signal peptidase. The proendothelin obtained is then processed by the protease furin on dibasic amino acid pairs to give a biologically inactive peptide comprising 38 amino acids, big endothelin (SEQ ID NO:3), from which finally the mature, biologically active endothelin-1 (SEQ ID NO:2) is formed by means of endothelin-converting enzymes (ECEs). Endothelin acts via the bond to specific receptors which are localized on muscle cells, myocytes and fibroblasts. This bond leads to efflux of calcium, activation of phospholipase C and inhibition of Na/K ATPase. In addition to the vasoconstrictive effect, endothelin also has growth-regulating properties.
In view of the detectable and presumably numerous and serious physiological effects of endothelins, in particular endothelin-1, various assays for its immunodiagnostic determination have been developed since the time of its identification and have been used for measurements of endothelin(s), in particular in human plasmas. The results of such determinations are the subject of numerous publications.
Raised plasma concentrations of endothelin-1 and big endothelin have been described for various clinical pictures [17]. These include cardiovascular diseases [1] (inter alia pulmonary hypertension [21], atherosclerosis [13], congestive heart failure [25], myocardial infarction [20]), sepsis and septic shock [11, 22, 23], cancer [2, 3, 15, 18], etc.
The immunoassays used for the measurements of endothelins in plasma samples (cf. the review in [17]) belonged in particular to the radioimmunoassay type (with marked endothelin-1 as competitor) or to the EIA/ELISA type and aimed exclusively at the determination of endothelin or the determination of an endothelin immunoreactivity. Assays of the RIA type have low specificity and also determine related peptides containing the endothelin sequence.
However, it was found that endothelin-1 (ET-1) has an extremely short residence time in the circulation and that it is removed from the circulation after only 1-2 min [6]. Since endothelin-1 in blood and plasma is considered to be stable [6], its distribution in other tissue and its rapid and high-affinity bonding to receptors are regarded as the most important reason for the short residence time. In certain tissues and body fluids, substantially higher endothelin-1 concentrations than, for example, in plasma could consequently be determined [1, 7]. In view of these circumstances, serious doubt was cast on the validity of the determination of ET-1 in plasma samples [17]. It is in fact to be assumed that the instantaneous ET-1 concentrations determinable in a plasma sample and reflecting in certain circumstances only a transition state are not important for the physiological effects of endothelin (ET-1) but that the sum of all free and bound, e.g. tissue- and receptor-bound, physiological ET-1 concentrations present in the organism are of much greater relevance.
The determination of the ET-1 precursor, of so-called big endothelin (“big ET-1”; SEQ ID NO:3), has the advantage over the determination of ET-1 that the residence time of “big ET-1” in the circulation is substantially longer than that of the ET-1 liberated therefrom. In a number of investigations, this “big endothelin” was therefore determined instead of the actual endothelin. In particular assays of the sandwich type which permit a reliable distinction of big endothelin-1 from processed ET-1 and other endothelins were used for its specific determination [4, 8, 10]. They showed that, in certain diseases, the increased ET immunoreactivities measured can be attributed to big ET.
The selective measurement of big-ET-1 represents only a gradual improvement but not an actual solution to the problem, since big endothelin too can be processed rapidly in blood circulation to give endothelin [1, 5, 9]. It therefore likewise has a relatively short biological half-life (20-25 minutes) [10], and consequently a measured value of the big endothelin determinable in the plasma likewise represents only an instantaneous plasma concentration and does not reflect the actual physiologically effective concentrations of endothelin. ET-1 formed physiologically under the conditions of a disease but already processed and bound into tissues or to receptors is not detected in plasma in the determination of big-ET-1. The total amount of physiologically active endothelin is therefore also underestimated in a measurement of big endothelin. An attempt to perform a supplementary specific measurement of the C-terminal peptide fragment of big-ET-1 (with the amino acids 74-90 of preproendothelin or the amino acids 20-38 of big endothelin) formed in the enzymatic cleavage of big-ET-1 in addition to ET-1 showed that this peptide is even less stable than ET-1 and is therefore unsuitable for measurements [10].
The prior art discloses only one commercial competitive test (N-terminal range 18-50, commercially available from Phoenix Pharmaceuticals; use for the sepsis diagnosis described in WO 00/22439) for evaluating ranges of proendothelin outside that of big endothelin. No information has been published regarding the stability and nature of the analyte to be evaluated using this assay.
It is the object of the present invention to develop an assay method which reflects the endogenous formation of big endothelin and endothelin, i.e. the total physiological concentration and hence action of endothelin, more reliably than the determinations to date of ET or big ET in plasma.
Such a method should be valid and capable of routine use and should be capable of providing reliable values for the physiological production of ET (ET-1) and/or its precursors in various pathological conditions, in particular in sepsis or other pathological conditions in which increased values for endothelin play a role.